Seed contamination with polyketide mycotoxins, including aflatoxin (AF) and sterigmatocystin (ST) produced by Aspergillus spp., is an agricultural, economic, and medical issue worldwide. Acetyl-CoA, the fundamental building block of all known fungal polyketides, is generated by a large number of biochemical pathways, including beta-oxidation of fatty acids and glycolysis of sugars. We present several lines of evidence to support a major role for seed fatty acids in formation of AF and ST in A. flavus, A. parasiticus, and A. nidulans. Aspergillus strains exhibiting canonical signs of oleic acid-induced peroxisome proliferation, including increased catalase activity, beta-oxidation gene expression, and peroxisomal clustering, also exhibited a marked increase in toxin gene expression and biosynthesis. Furthermore, microscopic observations showed that the ST and AF precursor norsolorinic acid accumulated in peroxisomes of all three Aspergilli. While a peroxisomal beta-oxidation mutation eliminated oleic acid-induced increases in ST in A. nidulans, a mitochondrial beta-oxidation mutation played a larger role in eliminating ST formation on oatmeal medium and on live corn kernels, implicating a fundamental role for both peroxisomal and mitochondrial beta-oxidation in toxin production.